The principal objective of this study is to elucidate metabolic functions of polyunsaturated fatty acids and phospholipids in nervous tissues with particular reference to their modulation by ethanol. In neuronal membranes, docosahexaenoic acid (22:6n3) is the most abundant polyunsaturated fatty acid esterified in phospholipids. Although 22:6n3 is thought to be essential for proper neuronal and retinal function, the exact mechanisms involved in its essentiality are not clearly understood. It is possible, however, that the release of 22:6n3 may play an important role in cell signaling as has been suggested for 20:4n6 hydrolyzed by cytosolic phospholipase A2 (PLA2). In order to test this possibility, we have examined the release of polyunsaturated fatty acids, 22:6n3 and 20:4n6 from neuronal membranes using rat brain synaptosomes and C6 glioma cells. Our study indicated that 20:4n6 was preferentially hydrolyzed from brain synaptosomes by endogenous lipase activity, despite the high abundance of 22:6n3 in synaptosomal membranes. This release was not blocked by inhibitors for low molecular weight PLA2 or diacylglycerol (DG) lipase, but blocked by cytosolic PLA2 inhibitors, suggesting that PLA2 activity may be primarily involved. Synaptosomal 22:6 species appeared to be resistant to hydrolysis even after stimulation with various agonists, suggesting that 22:6n3 as a membrane component rather than as a released free fatty acid may be of more physiological importance. Similar results were obtained for primary cultures of rat hippocampal neurons. In contrast to the synaptosomal lipase activity, glioma cells and primary cultures of rat astroglia hydrolyzed 20:4n6 and 22:6n3 equally well under both stimulated and unstimulated conditions, indicating a lack of specificity. Facilitated release of 22:6n3 from glia cells may indicate that one of the supporting roles of astroglia may be providing 22:6n3 to neuronal membranes.